TWI290226B - Method for detecting human HCCR-1 protein and a kit for diagnosing liver cancer using the same - Google Patents
Method for detecting human HCCR-1 protein and a kit for diagnosing liver cancer using the same Download PDFInfo
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1290226 玖、發明說明: 【發明所屬之技術領域】 本發明係有關一種經由使用可特異地結合到從人類原_致癌基因 表現出的蛋白質之抗體進行抗原,抗體結合反應以偵檢一樣品内所含人類 HCCR-1蛋白質之方法,與一種使用彼診斷肝癌所用之套組。 【先前技術】 一般而言,為了增加肝癌患者的存活率,最重要者為在早期經由讓具有高 度罹患肝癌危險率的人到醫院定期健康檢驗以偵檢出Hcc (肝細胞癌) (Hepatocellularcarcinoma)的存在。具有高度罹患肝癌風險率的人稱為,,高肝 癌危險組’’。與肝癌相關聯的危險因素大部分經分類為生理學因素與病理學 因素。生理學因素的例子包括年齡、性別、種族等,而病理學因素的例子 包括B型肝炎病毒或C型肝炎病毒感染,肝硬化,酒精濫用,暴露於黃麴 毒素(aflatoxin),相當罕見的新陳代謝疾病,性激素,化學物質等。 到目前為止,根據正常成人血清中的AFP濃度幾乎為零但是在許多患有肝 癌的患者體内卻劇烈增加之發現而將〇;_胎蛋白水平有利地用於肝癌早期偵 檢的診斷中作為諸篩選因素之一。不過,事實上,MP的濃度不僅會在肝 癌中增加而且也會在良性疾病,例如慢性肝炎或肝硬化,惡性疾病,例如 絨毛膜癌(choriocarcinoma)或肝母細胞瘤(hepatoblastoma),或懷孕等之中增 加。相反地,經報導AFP不能有利地用於肝癌的早期診斷,如指出早期hcc 血清中的AFP濃度都呈實質地正常水平之許多臨床實驗所揭露出者 (Collier J and Sherman M? Hepatology 27:273-278, 1998; Okuda K5 J. Hepatol 32(1 Suppl):225-237, 2000)。根據此報告,於某些患有慢性肝炎或肝硬化但 1290226 沒有HCC的患者經觀察出血清細水平有非特別地增加。而且,大約篇 HCCs患者展現出正常AFP水平,且超過8〇%具有直徑不大於2厘米的小 HCC之患者顯示出在AFP水平上的統計無_差異,使得鮮與正常成人 所具者實質地不能相比。 有關HCC患者體_ AFP水平是否適合祕賴辑巾,有數份先前報告 可以提及如τ。有-純告歧㈣46.2%m患直财狀5 _的hccs 之患者_的AFP水平係實質地正常者。有另—份報絲示出於約 25.5% 罹患直徑不大於5厘米的HCCs之患者_之App水平係實f地正常者而 於約34.8%罹患不大於3厘米的相當小直徑HCCs之患者體内展現出正常 AFP 水平(Collier J and Sherman M,//epato/og); 27: 273_278, 1998; κ,』 /fepato/· 32(1 Suppl): 225-237, 2000)。因此等理由之故,雖然Αρρ檢驗經認 為可以到某種程度地作為在慢性肝病患者中篩選出HCCs患者之必需步 驟,不過以AFP水平為基礎的肝癌診斷需要許多考慮事項。因此,有需要 發展出一種新的血清學檢驗,其比傳統AFP檢驗具有遠較為高的敏感度和 特異性。 有關一部分此種研究,最近發現依照AFP二醇系列構造(giycol_series structures)的多樣亞型而定,外源凝集素-反應性部分(lectin_reactive fracti〇ns) 會有所不同。根據AFP-L3部分為對肝癌較具特異性的亞型之發現,有一份 報告述及可以經由測定AFP-L3部分作出比習用的App檢驗對肝癌更具特 異性之診斷檢驗(Okuno M et al·,』G如咖她ra/./^7偷/. 16(12):1329_1335, 2001)。不過,由於與臨床診斷相關的數項限制,此種嘗試不能得到作為有 用的肝癌疹斷工具之效效用性。 1290226 另一種可能的診斷檢驗為超聲波篩選,其可用簡單,安全的方式實施。不 過,此種方法具有不良的預測能力,轉而不能偵檢直徑小於丨厘米的微小 病變。而且,由於此種技術有賴於檢驗員的技術或經驗,因此在診斷正確 性上可能導致根據檢驗員所致頗為不同的結果,可推測出其缺乏客觀性 (objectivity)。再者,頗難以將超聲波應用於患有嚴重肝硬化徵候且具有高 度肝癌危險性的患者,使其實質地不能準確地鑑別出病變。 所以’為了達浙癌的早期賴財效_測,有高度需要發展出一種新 的’具有改良的敏感度和特異性之血清學檢驗。 綜上所述,為了發展出_種有效的肝癌早期診斷的檢驗方法,本案發 明人乃進行精心探討研究且鑑別出經由使用可特異地結合到從人類原致癌 基因HCCR-1表現出的蛋白質之抗體進行抗原_抗體結合反應可以有效地偏 檢出肝癌,人類原-致癌基因HCCm經寄存於知,登錄號碼 AF1956卜如韓國專利公報第2〇〇1_39556號中所述者。此項發現導致本案 發明人完成本發明。 【發明内容】 為了解决上述制題’本判的—項目的為提出—種使用人麵致癌基因 HCCR 1有效地在早期階段偵檢職之方法與—種使用彼診斷肝癌所用之 套組。 為了凡成上述本發崎目的,提出—種經由使用可特異地結合到從人類原-致癌基因HCCR_1表現㈣蛋白f之抗舰行抗原_抗體結合反應而測量一 樣品的活體内(in咖赋叫蛋自質表現水平以診斷肝癌之方法,與一種 1290226 使用彼診斷肝癌所用之套組。 【圖式簡單說明】 圖1圖解祝雜縣發賢從獅到ρΜΑ£·ρ;2χ/Ηα^_ι雜内的大腸桿 菌(Escherichia coli (Ε· coli)) BL21菌株離析和純化出之HCCR_i重組蛋白 實施免疫點潰的結果; 圖2圖解說明根據本發明制HCCR]重組蛋白作為抗原實施肌财以筛 選會產生HCCR-1早株抗體的融合瘤細胞系之結果; 圖3圖解說明根據本發明使用配❿丨單株抗體對Hcc組織實施免疫檢定 之結果,其中A和B顯示出者為HCC組織的免疫檢定結果,c顯示出肝炎 組織的免疫檢定結果,且D顯示出正常肝組織的免疫檢定結果;且 圖4a至4e圖解說明根據本發明以使用HCCR_i單株抗體的診斷套組偵檢 HCC和肝炎患者的血清,母體血清和正常血清中hccrj蛋白質的表現所 得肝癌診斷結果,其中圖4a顯示出得自HCC患者的血清之吸光度, 圖4b顯示出得自肝炎患者的血清之吸光度,圖4c顯示出母體血清的吸光 度’圖4c顯示出正常血清的吸光度,且圖4e顯示出根據從標準曲線截止值 對個別樣品的吸光度水平所做比較之結果。 【實施方式】 後文中,要參照所附圖式詳細說明本發明具體實例。 人類原-致癌基因HCCR-1 (GenBank Accession No· AF195651;韓國專利公 報第2001-39556號),其係位於第12號染色體(Chromosome No. 12 的長臂之中且具有開放編閱架構(0Penreading frame),其再哺乳動物體内過 1290226 度表現(overexpressed)之時,具有成為致癌基因之作用,且從該基因會衍生 出具有約4〇kDa的大小之蛋白質(稱為HCCR]蛋白質)。 HCCR_1蛋白質特異性抗體較佳者係從經由在動物體内免疫化抗 原蛋白質所得抗血清純化出。更佳者,從經由在母雞體内免疫化 HCCR-1 蛋白貝所知血清或蛋純化出Ηα:ΙΜ抗原蛋白質特異性抗體。蛋 白質特異性抗體可為多株抗體或為單株抗體。 要合成可特鏡_ HCOM蛋白f之抗體,魏要取得hccim蛋白 質。HCCR-1蛋白質可以經由使記知的胺基酸序列予以合成或經由基因 工程法製造成為纽蛋自麵。例如,可以使肖包括下列轉的方法進行 製備:使用在the NIH program GenBank資料庫中所列的hccr]基因之 驗序列製備出可魏丨呈纽蛋白形式的蛋㈣之重組 蛋白的表現载體;經由將表現載體轉形到大腸桿菌_以得到要產生 HCCR 1重組蛋白所用的轉形體;及培養該轉形體以離析/純化出人類原_ 致癌基因HCCR-1重組蛋白。1290226 玖Invention Description: [Technical Field] The present invention relates to an antigen-antibody binding reaction for detecting a sample by using an antibody that specifically binds to a protein expressed from a human proto-oncogene A method comprising a human HCCR-1 protein, and a kit for use in diagnosing liver cancer. [Prior Art] In general, in order to increase the survival rate of patients with liver cancer, the most important one is to detect Hcc (hepatocellular carcinoma) in the early stage by letting people with high risk of liver cancer go to the hospital for regular health tests. The presence. A person with a high risk of developing liver cancer is called a high-hepatic cancer risk group. Most of the risk factors associated with liver cancer are classified into physiological factors and pathological factors. Examples of physiological factors include age, gender, ethnicity, etc., and examples of pathological factors include hepatitis B virus or hepatitis C virus infection, cirrhosis, alcohol abuse, exposure to aflatoxin, and relatively rare metabolism. Diseases, sex hormones, chemicals, etc. So far, according to the fact that the concentration of AFP in normal adult serum is almost zero, but it is found in many patients with liver cancer, the sputum is increased; the fetal protein level is favorably used in the diagnosis of early detection of liver cancer. One of the screening factors. However, in fact, the concentration of MP will not only increase in liver cancer but also in benign diseases such as chronic hepatitis or cirrhosis, malignant diseases such as choriocarcinoma or hepatoblastoma, or pregnancy, etc. Increased among them. Conversely, it has been reported that AFP cannot be advantageously used for the early diagnosis of liver cancer, as indicated by many clinical trials that indicate that AFP concentrations in early hcc sera are substantially normal (Collier J and Sherman M? Hepatology 27:273 -278, 1998; Okuda K5 J. Hepatol 32 (1 Suppl): 225-237, 2000). According to this report, there was a non-specific increase in serum fine levels observed in some patients with chronic hepatitis or cirrhosis but 1290226 without HCC. Moreover, approximately HCCs patients exhibit normal AFP levels, and more than 8% of patients with small HCCs with a diameter of no more than 2 cm show no statistical difference in AFP levels, making fresh and normal adults substantially Can't compare. Regarding whether the HCC patient's body _ AFP level is suitable for the secret, there are several previous reports that can be mentioned as τ. There are - purely disambiguated (four) 46.2% m patients with a direct financial situation of 5 _ hccs _ AFP levels are substantially normal. Another report showed that about 25.5% of patients with HCCs with a diameter of no more than 5 cm had an App level that was normal and about 34.8% of patients with a relatively small diameter HCC of no more than 3 cm. The normal AFP levels are exhibited (Collier J and Sherman M, // epiato/og); 27: 273_278, 1998; κ, 』 /fepato/· 32 (1 Suppl): 225-237, 2000). For this reason, although the Αρρ test is considered to be a necessary step to select a patient for HCCs in patients with chronic liver disease to some extent, the diagnosis of liver cancer based on AFP levels requires many considerations. Therefore, there is a need to develop a new serological test that has far greater sensitivity and specificity than traditional AFP testing. Some of these studies have recently found that the lectin_reactive fracti〇ns will vary depending on the various subtypes of the AFP diol series structure (giycol_series structures). According to the findings of the subtypes of AFP-L3 that are more specific for liver cancer, a report mentions that a more specific diagnostic test for liver cancer can be made by measuring the AFP-L3 part of the App test (Okuno M et al ·, 』G as a coffee she ra /. / ^ 7 steal /. 16 (12): 1329_1335, 2001). However, due to several limitations associated with clinical diagnosis, such an attempt cannot be used as a useful tool for the use of a liver cancer rupture tool. 1290226 Another possible diagnostic test is ultrasound screening, which can be implemented in a simple, safe manner. However, this method has poor predictive ability and cannot detect small lesions smaller than 丨 cm in diameter. Moreover, since this technique relies on the inspector's skill or experience, it may lead to quite different results based on the inspector in terms of diagnostic accuracy, and it can be inferred that it lacks objectivity. Furthermore, it is quite difficult to apply ultrasound to patients suffering from severe cirrhosis and having a high risk of liver cancer, so that it is substantially incapable of accurately identifying the lesion. Therefore, in order to achieve early detection of cancer, it is highly desirable to develop a new serological test with improved sensitivity and specificity. In summary, in order to develop an effective test method for early diagnosis of liver cancer, the inventors of the present invention have carefully studied and identified the use of proteins that specifically bind to human protooncogene HCCR-1. The antigen-antibody-binding reaction of the antibody can effectively detect the liver cancer, and the human pro-oncogene HCCm is deposited in the same name as that described in Korean Patent Publication No. 2-39556. This finding led the inventor of the present invention to complete the present invention. SUMMARY OF THE INVENTION In order to solve the above-mentioned problem, the present invention proposes a method for effectively detecting an occupational oncogene HCCR 1 at an early stage and using a kit for diagnosing liver cancer. For the purpose of the above-mentioned present, it is proposed to measure a sample in vivo by using an anti-shipping antigen-antibody binding reaction which specifically binds to the human pro-oncogene HCCR_1 (4) protein f (in The method of diagnosing liver cancer is called the method of diagnosing liver cancer, and a set of 1290226 used for diagnosing liver cancer. [Simple diagram of the diagram] Figure 1 illustrates the sage of xian xian from lion to ρΜΑ£·ρ; 2χ/Ηα^ Escherichia coli (E. coli) BL21 strain isolated and purified HCCR_i recombinant protein to perform the results of immunospotting; Figure 2 illustrates the production of HCCR] recombinant protein as an antigen according to the present invention Screening results for fusion cell lines that produce HCCR-1 early strain antibodies; Figure 3 illustrates the results of immunoassays for Hcc tissues using a sputum monoclonal antibody in accordance with the present invention, wherein A and B are shown as HCC The results of the immunoassay of the tissue, c shows the results of the immunoassay of the hepatitis tissue, and D shows the results of the immunoassay of the normal liver tissue; and Figures 4a to 4e illustrate the use of the HCCR_i single according to the present invention. The diagnostic kit of antibodies detects the serum of HCC and hepatitis patients, the results of liver cancer diagnosis of hccrj protein in maternal serum and normal serum, wherein Figure 4a shows the absorbance of serum from HCC patients, and Figure 4b shows the results from hepatitis. The absorbance of the patient's serum, Figure 4c shows the absorbance of the maternal serum' Figure 4c shows the absorbance of normal serum, and Figure 4e shows the results of the comparison of the absorbance levels of the individual samples from the cutoff value of the standard curve. Hereinafter, specific examples of the present invention will be described in detail with reference to the accompanying drawings. Human proto-oncogene HCCR-1 (GenBank Accession No. AF195651; Korean Patent Publication No. 2001-39556), which is located on chromosome 12 ( The long arm of Chromosome No. 12 has an open editing frame (0Penreading frame), which has the function of becoming an oncogene when it is overexpressed in the mammalian body, and is derived from the gene. A protein having a size of about 4〇kDa (referred to as HCCR) protein. HCCR_1 protein-specific antibody is preferred The antiserum obtained by immunizing the antigenic protein in the animal is purified. More preferably, the Ηα: ΙΜ antigen protein-specific antibody is purified from the serum or egg known by immunizing HCCR-1 protein in the hen. The antibody may be a plurality of antibodies or a monoclonal antibody. To synthesize an antibody of HCOM protein f, we need to obtain hccim protein. HCCR-1 protein can be synthesized by genetic acid engineering or genetic engineering. The law is made into a new egg. For example, Sha can be prepared by the following method: using the hccr] gene sequence listed in the NIH program GenBank database to prepare a recombinant protein expression vector of the egg (4) The human proto-oncogene HCCR-1 recombinant protein is isolated/purified by transforming the expression vector into E. coli to obtain a transformant for producing the HCCR 1 recombinant protein; and culturing the transformant.
於本發明-較佳具體實例中,係經由轉形到含有hccr心基因的 pMALp2X/HCCR_l紐之大腸桿_ blu產^具有、麵合到队端的麥芽 糖之重組蛋白,接著用恰當的酵素離析/純化以移轉麥芽糖之既CR_1蛋 白質以作為抗原蛋白質。為了鑑定出經如此產生的蛋白質為-種HCCIU 重、且蛋白乃用西方氏點潰分析(Western blot analysis)觀察具有分子量約% kDa的HCCR-1重組蛋白之特異性谓檢。 使用從大鱗轉频騎且純化㈣纽蛋自作為抗原以筛 選與分析可以經由小鼠的免疫化與細胞融合產生單株抗體的融合瘤細胞 1290226 系。 發展出融合瘤細胞系所需的免疫化小鼠係經由將HCCR_1重組蛋白與一相 當量的Freimd,S完全佐劑充分混合直到混合物乳化為止再將其經由腹膜内 注射到小鼠體内,接著加強注射以增加小鼠的免疫原性而產生的。較佳者, 後續加強注射只用HCCR-1重組蛋白經由腹膜内途徑投到小鼠三次而不使 用佐劑。 收集透過注射HCCR-1重組蛋白抗原予以免疫化的小鼠血清且測量抗體滴 定度。 將形成抗體的小鼠之脾細胞與細胞融合的母細胞以1〇:丨的細胞數目比例 混合且接著在聚乙二醇内融合,然後添加培養基予以稀釋。然後,將融合 瘤細胞轉移到篩選培養基,且隨即予以培養。於清楚地觀察到融合瘤細胞 組的生長之時,將培養基更換成篩選培養基策進細胞的增生。可用的細胞 融合母細胞之例子包括SP2/0 Agl4細胞系,P3x653細胞系,和NS-1細 胞系。可以使用的培養基之例子包括PMI培養基,dmem培養基,imdm 培養基,和F12培養基。可用的篩選培養基之例子包括jjat培養基。 於在篩選培養基内生長的諸融合瘤細胞組中,以間接酵素聯結免疫吸著檢 定(mdifeet enzyme_linked immunosorbant assay) (ELISA)法只篩選出能夠與 HCCR-1蛋白質抗原特異地反應的融合瘤細胞組別。換言之,以eusa讀 取器測量吸光度以篩選會分泌出對經純化出的HCCR4蛋白質具有高度結 合性之抗體的融合瘤細胞系,經由限制性稀釋將融合瘤細胞稀釋以使篩選 出的細胞成為單株系,藉此建立由從一單一細胞增生出的細胞所構成的細 胞系。 1290226 對人類原姻基因HCCW蛋白質財類性之單株抗射峨用已建立 的細胞系透過下列諸步驟大量產生: 1) 將融合瘤細胞系經由腹膜内途徑注射到小氣體内; 2) 從小鼠的肥大腹部收集腹水液;及 3) 從腹水液騎㈣HCQM蛋自質具树異性之單株抗體蛋白質。 經由使用如此產生的HCOM特異性抗白㈣行抗原_抗體結合反應 以診斷癌症可以經由測定該蛋白質在取自一檢體的一樣品内之活體内表現 於以完成。表現水平可以用技藝中已知的技術予以舰,包括酵素聯結免籲 疫吸著檢定(ELISA>,放射免疫檢定(RIA),夾層檢定,和在聚丙稀酿胺凝 膠上進行的西方氏點潰或免疫點潰(immun〇bl〇tting)分析。 於-較佳具體實例中,係透過下列諸步驟使用Ηα:ΙΜ蛋白質特異性抗體 進行ELISA技術: 1) 將-檢體和-對照組樣品放到一塗覆著Η(:αΜ特異性抗體的反應器内 以誘發抗原-抗體反應; 2) 使用繼發性抗體-標記拼合物(sec〇ndary antib〇dy撼d⑽_ 兄物質的顏色顯現受質溶液偵檢抗原―抗體反應產物;及 3) 比較檢體的偵檢結果與對照組的結果。 本發明也提出-蛋白質晶片,其可以經由將HCCW特異性抗體蛋白質固 定在-生物微晶片之上’促成彼與收集自一個體的活體内樣品之間的反應 而保漢該HCCR-1特異性抗體蛋白質的抗原。使用已知技術,例如eLisa、 生物微晶片或自動微陣列系統,可以分析大量的樣品。 另外,本發明提供一種用以診斷肝癌之套組,其包括可與HCCR4特異地 11 1290226 反應之抗體,藉此促成肝癌的早期診斷。 本發明診斷套組包括: 1) 一經塗覆著HCCR-1特異性抗體的反應器; 2) 含有一 HCCR-1標準抗原的陽性對照組與一含有於其體内注射入該抗 原的動物之抗血清的陰性對照組; 3) 龜發性抗體拼合物,其具有一經拼合的標記物質,該標記物質可以經 由與一受質反應而產生顏色; 4) 一顏色顯現性受質溶液,可與該標記物質反應以產生顏色; 5) —用於每一步驟中的洗滌溶液;與 6) 一酵素反應停止溶液。 該W斷套組可以經由抗原_抗體結合反應定量地或定性地分析抗體蛋白質的 抗原而診斷肝癌。該抗原_抗體結合反應可以用通常為技藝中已知的技術予 以偵檢,包括ELISA,RIA,夾層檢定,在聚丙烯醯胺凝膠上的西方氏點 /貝,與免疫點潰分析。例如,可以提供該診斷套組以供使用塗覆著重組單 株抗體蛋白質的96-洞微滴定板之ELISA所用。 了 乂用來在其上面塗覆抗體蛋白質的反應器之例子包括硝酸纖維素膜,聚 乙烯樹脂製造成的96_洞板,聚苯乙烯樹脂製造成的96_洞板,和載玻璃。 如上面所述者’根據本發明的抗體較佳者係從在一動物體内免疫化 抗原蛋白質所得抗血清純化出者。HCCRq蛋白質特異性抗體較佳者係對 母反應器以約1至1〇微克/lQ〇微升的比率塗覆。 在根據本發明的診斷套組中所含對照組包括陽性對照組和陰性對照組。陽 Μ十,展組為含有HCCR-1蛋白質標準抗原的混合物,且陰性對照組為沒有 12 1290226 感染到HCCR-1蛋白質抗原的動物血清。於本發明實施例中,係使用具有 多種蛋白質濃度:0毫微克/毫升(ng/ml) (A),2〇毫微克/毫升(B),4〇毫微 克/毫升(C),80毫微克/毫升(D),160毫微克/毫升(E),320毫微克/毫升(F) 和640毫微克/毫升(G)之HCCR_1蛋白質標準抗原溶液。 有關繼發性抗體標記物質’較佳者為使用已知的可誘導顏色顯現的顏色顯 現劑,且可用於本發明中的標記物質之例子包括辣根過氧化酶(HRp),鹼 性磷酸酶,膠體金,螢光素例如聚L_離胺酸_螢光素異硫氰酸酯(p〇ly L_lysine_fluorescein iS0thi0cyanate) (FITC)或若丹明办異硫氰酸酯 (rh〇damine-B_is〇thi〇Cyanate) (RITC),和染料。於本發明中,係使用,例如, 山羊抗-兔子 IgG-HRP 拼合物(g0at anti_rabbit c〇njugate) (IgG HRp 拼合物)。 較佳者,所使用的顏色顯現受質係根據顏色顯現所包括的標記物質而變 異,且其可用的例子包括3,3,,5,5,_四甲基聯苯胺(3,3,,5,5,擔_邮^述叫 (TMB) ’ 2,2’-井基-雙(3_乙基苯并噻唑啉 -6-磺酸) (2,2 ·&ζίηο-1^(3_6%^ηζ(Λΐώζο1ή^_6-3ΐι1ίόιήο acid) (ABTS),與鄰申苯二胺 (0PD)。更佳者,顏色顯現受質係以溶解在一緩衝溶液(〇丨Μ 内的狀態提供。該顏色顯現受質例如TMB係經用為繼發性抗體拼合物的 標記物質之所分解HRP以產生一顏色顯現沉澱物。顏色顯現沉殿物的沉 澱水平係用肉眼觀察,藉此測定HCCR4蛋白質抗原之存在。 該洗滌溶液較佳地包括磷酸鹽緩衝液,Naa,和Tween 2〇,更佳者為含有 〇·〇2 Μ磷酸鹽緩衝液,〇·13 M NaC1,和〇 〇5% Tween 2〇之緩衝溶液。於抗 原-抗體結合反應,與抗原-抗體拼合物上的繼發性抗體反應之後,將一恰當 13 1290226 量的洗餘液供給到反應H内。用絲溶液進行的洗難經重複地實施3 到6次。較佳者係使用含G_1% BSA的磷酸鹽緩衡液作為阻斷溶液且較佳 者使用2N硫酸溶液作為酵素反應停止溶液。 至此要·制本發曝斷套組傭在_檢雌品内的抗原以在 早期階段麟肝狀松。將HOW單魏體分顺在雜和陰性對照 組内的檢雜品反應,抑絲溶液絲。紐,於所得録巾加入經用 可與受質反縫生顏色的標記物f標記著之繼發餘簡合物且再使用洗 務溶液洗蘇。織,於所得產物中加人含有受f的溶液以誘導顏色顯現。 然後’測量在4〇5奈米的吸光度。此處,標準抗原溶液A的平均吸光度係 大於或等於0·_到、於鱗於綱。標準抗縣液f的平均吸光度係大 於或等於1.200且小於或等於3._。標準抗原溶液A和f的吸光度之間的 平均值伽奴為-餘似祕後絲狀㈣為雜減或陰性檢 體。當檢_吸錢大於鮮抗絲液F的吸歧之時,_檢體稀釋且 再度測1其吸光度。具有高域止_吸光度之檢斷鑑定為陽性,而具 有低於截止值的吸光度之檢體即鑑定為陰性。 經確定者根據本個含有人__致絲因Ηα:]Μ特紐抗_肝癌診斷 套組,為-驗㈣者血相卿免鮮輯卫具,具有轉統聊檢 驗工具更高的準確度和再現性(reproducibility)。而且根據本發日月的診斷套 組能夠對即使是AFP陰性的檢體細綺癌畴在。細言之,根據本發 明的肝癌早__⑽輯學腦^檢義—約Μ⑽的診斷正確 性’此在統計有效性上高於傳統AFP檢驗。而且,即使對於卿陰性的 肝癌組織,也可得到約93.1%_著地較為高之偵檢率。再者,根據本發 14 1290226 明的HCCR_1診斷對於直徑不大於2厘米的小Hcc檢體也比傳統App診 斷顯示出約91.7%的相對較高之診斷績效。亦即,於使用根據本發明的 HCCR_1以免疫染著法染著正常肝組織,硬化組織和HCC組織之時,於正 常肝組織與肝硬化組織中沒有偵檢到會與HCCR4反應的抗原,而在只有 在HCC組織中才偵檢到會與HCCR-1反應的抗原,展現出深棕色的顯現。 所以,根據本發明的診斷方法與套組可以非常有利地用於肝癌的早期診斷 與小HCC的診斷,因為彼等相對於傳統App檢驗具有高準確度與再現性之 至此要藉由下面的實施例詳細闡明本發明,但是絕不將本發明範圍限制到 諸實施例中所述特定具體實例。 <實施例1>HCCR_1多株抗體之產生 <1-1> HCCR-1重組蛋白之產生 為了產生人類原-致癌基因HCCR-丨特異性抗體作為肝癌診斷所用的腫瘤標 記性物質,乃將部分人類原致癌基因(對胁GenBank &⑽i〇n n〇 AF195651 的驗序歹,jID.N〇s: 123_473,胺基酸4mDN〇s: 39i5置於 pET-32(+)載In a preferred embodiment of the present invention, a recombinant protein having a maltose having a face-to-team end, which is transformed into a large intestine rod _blu containing pmcp2X/HCCR_l containing a hccr heart gene, is then isolated by an appropriate enzyme/ Purification to transfer both the CR_1 protein of maltose as an antigenic protein. In order to identify the protein thus produced as a species of HCCIU heavy, and the protein was observed by Western blot analysis, the specificity of the HCCR-1 recombinant protein having a molecular weight of about % kDa was observed. The fusion tumor cell 1290226 line which produces monoclonal antibodies by immunization and cell fusion of mice by screening and analyzing from the scales of the large scales and purifying (4) eggs is used as an antigen. The immunized mouse line required to develop a fusion tumor cell line is thoroughly mixed with a considerable amount of Freimd, S complete adjuvant until the mixture is emulsified, and then intraperitoneally injected into the mouse, followed by intraperitoneal injection into the mouse. Intensive injections are produced to increase the immunogenicity of mice. Preferably, subsequent booster injections are administered to the mice three times via the intraperitoneal route using only HCCR-1 recombinant protein without the use of an adjuvant. Mouse sera immunized by injection of HCCR-1 recombinant protein antigen were collected and antibody titers were measured. The spleen cells of the antibody-forming mouse spleen cells were mixed with the cells in a ratio of the number of cells of 〇: 且 and then fused in polyethylene glycol, and then the medium was added for dilution. The fused tumor cells are then transferred to a screening medium and cultured immediately. At the time when the growth of the fusion tumor cell group was clearly observed, the medium was changed to a selection medium to promote cell proliferation. Useful cells Examples of the fusion mother cells include the SP2/0 Agl4 cell line, the P3x653 cell line, and the NS-1 cell line. Examples of the medium which can be used include PMI medium, dmem medium, imdm medium, and F12 medium. Examples of useful screening media include jjat media. In the fusion tumor cell group grown in the screening medium, only the fusion tumor cell group capable of specifically reacting with the HCCR-1 protein antigen was selected by the mdifeet enzyme-linked immunosorbant assay (ELISA). do not. In other words, the absorbance is measured with an eusa reader to screen for a fusion tumor cell line that secretes antibodies that are highly binding to the purified HCCR4 protein, and the fusion tumor cells are diluted by limiting dilution to make the screened cells single. A strain whereby a cell line composed of cells proliferating from a single cell is established. 1290226 The individual cell line of the human calamity gene HCCW protein is used to produce a large number of cell lines through the following steps: 1) Inject the fusion cell line into the small gas via the intraperitoneal route; 2) From small Rat's hypertrophied abdomen collects ascites fluid; and 3) rides from ascites fluid (IV) HCQM egg self-quality tree-specific antibody protein. The diagnosis of cancer by using the HCOM-specific anti-white (four) antigen-antibody binding reaction thus produced can be accomplished by measuring the in vivo expression of the protein in a sample taken from a sample. Performance levels can be obtained using techniques known in the art, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIA), sandwich assays, and Western points on polypropylene gels. Analysis of the ulceration or immunization bl〇tting. In a preferred embodiment, the ELISA technique is performed using the Ηα:ΙΜ protein-specific antibody by the following steps: 1) The sample and the control sample are Placed in a reactor coated with hydrazine (:αΜ specific antibody to induce antigen-antibody reaction; 2) using secondary antibody-labeled complex (sec〇ndary antib〇dy撼d(10)_ Qualitative solution detection antigen-antibody reaction product; and 3) comparison of the detection results of the sample with the results of the control group. The present invention also proposes a protein wafer that can protect the HCCR-1 specificity by immobilizing the HCCW-specific antibody protein on the biomicrochip to facilitate the reaction between the sample and the in vivo sample collected from one body. The antigen of the antibody protein. A large number of samples can be analyzed using known techniques, such as eLisa, biomicrochips or automated microarray systems. Further, the present invention provides a kit for diagnosing liver cancer comprising an antibody reactive with HCCR4 specifically 11 1290226, thereby facilitating early diagnosis of liver cancer. The diagnostic kit of the present invention comprises: 1) a reactor coated with an HCCR-1 specific antibody; 2) a positive control group containing a HCCR-1 standard antigen and an animal containing the antigen injected therein a negative control group of antiserum; 3) a tortoise-like antibody composition having a flattened labeling substance which can react with a substrate to produce a color; 4) a color developing substrate solution, The labeling substance reacts to produce a color; 5) - for the washing solution in each step; and 6) an enzyme to stop the solution. The W-cut kit can diagnose liver cancer by quantitatively or qualitatively analyzing the antigen of the antibody protein via an antigen-antibody binding reaction. The antigen-antibody binding reaction can be detected by techniques commonly known in the art, including ELISA, RIA, sandwich assay, Western Point/Bei on polyacrylamide gel, and immunospot analysis. For example, the diagnostic kit can be provided for use in an ELISA using a 96-well microtiter plate coated with recombinant monoclonal antibody protein. Examples of the reactor for coating the antibody protein thereon include a nitrocellulose membrane, a 96-hole plate made of a polyethylene resin, a 96-hole plate made of a polystyrene resin, and a carrier glass. As described above, the antibody according to the present invention is preferably purified from an antiserum obtained by immunizing an antigenic protein in an animal. The HCCRq protein-specific antibody is preferably applied to the parent reactor at a ratio of about 1 to 1 μg/lQ〇 microliter. The control group contained in the diagnostic kit according to the present invention includes a positive control group and a negative control group. Yang Μ 10, the exhibition group is a mixture containing the standard antigen of HCCR-1 protein, and the negative control group is the serum of the animal which does not have 12 1290226 infected with HCCR-1 protein antigen. In the examples of the present invention, a plurality of protein concentrations are used: 0 ng/ml (ng/ml) (A), 2 ng/g (B), 4 ng/g (C), 80 m. Micrograms/ml (D), 160 ng/ml (E), 320 ng/ml (F) and 640 ng/ml (G) of HCCR_1 protein standard antigen solution. The secondary antibody-labeling substance is preferably a color developing agent using known inducible color development, and examples of the labeling substance usable in the present invention include horseradish peroxidase (HRp), alkaline phosphatase. , colloidal gold, luciferin such as poly-L-lysine _ fluorescein isothiocyanate (p〇ly L_lysine_fluorescein iS0thi0cyanate) (FITC) or rhodamine isothiocyanate (rh〇damine-B_is〇 Thi〇Cyanate) (RITC), and dyes. In the present invention, for example, a goat anti-rabbit IgG-HRP complex (g0at anti_rabbit c〇njugate) (IgG HRp composition) is used. Preferably, the color appearance used varies depending on the labeling substance included in the color development, and useful examples thereof include 3,3,5,5,4-tetramethylbenzidine (3,3,, 5,5, 担_邮^说(TMB) ' 2,2'-well base-bis(3_ethylbenzothiazoline-6-sulfonic acid) (2,2 ·&ζίηο-1^( 3_6%^ηζ(Λΐώζο1ή^_6-3ΐι1ίόιήο acid) (ABTS), with o-phenylenediamine (0PD). More preferably, the color appears to be supplied by the matrix to dissolve in a buffer solution (in the state of 〇丨Μ). This color develops a substance such as TMB which is decomposed by the HRP which is used as a labeling substance of the secondary antibody composition to produce a color-developing precipitate. The color level of the precipitate is observed by the naked eye, thereby measuring the HCCR4 protein. The presence of the antigen. The washing solution preferably comprises phosphate buffer, Naa, and Tween 2, more preferably 〇·〇2 Μ phosphate buffer, 〇·13 M NaC1, and 〇〇5% Tween 2〇 buffer solution. After the antigen-antibody binding reaction, after reacting with the secondary antibody on the antigen-antibody complex, an appropriate amount of 13 1290226 will be The raffinate is supplied to the reaction H. The washing with the silk solution is repeated 3 to 6 times. Preferably, a phosphate buffer containing G_1% BSA is used as a blocking solution and preferably 2N is used. The sulfuric acid solution is used as an enzyme reaction stop solution. At this point, the antigen in the female product is used to detect the antigen in the female product at an early stage. The HOW single Wei body is divided into the heterozygous and negative control groups. The reaction of the impurity is detected, and the silk solution is added. In the obtained recording towel, the secondary residue which is marked with the label f which can be colored with the reverse stitching color is added, and the washing solution is used for washing. Adding a solution containing f to the obtained product to induce color development. Then 'measuring the absorbance at 4〇5 nm. Here, the average absorbance of the standard antigen solution A is greater than or equal to 0·_ to the scale Yu Gang. The average absorbance of the standard anti-county liquid f is greater than or equal to 1.200 and less than or equal to 3._. The average value between the absorbances of the standard antigen solutions A and f is - the rest is like the filament (4) is miscellaneous Reduced or negative sample. When the test _ sucking money is greater than the absorption of fresh anti-filament F, _ Body dilution and re-measurement of its absorbance. The detection with high domain stop_absorbance is identified as positive, while the sample with absorbance below the cutoff value is identified as negative. The determined person is based on this person. Because Ηα:] Μ特纽抗 _ liver cancer diagnosis kit, for the test (four) of the blood phase of the liberation of the guardian, with a higher accuracy and reproducibility of the test tool. The monthly diagnostic kit can be used for even the AFP-negative specimens. In summary, the diagnosis of liver cancer according to the present invention is __(10) and the diagnostic accuracy of 脑(10) is statistically effective. Sexually higher than the traditional AFP test. Moreover, even for the liver cancer tissues with negative results, a detection rate of about 93.1% _ is relatively high. Furthermore, the HCCR_1 diagnostic according to the present invention has a relatively high diagnostic performance of about 91.7% for small Hcc specimens having a diameter of no more than 2 cm than conventional App diagnostics. That is, when the normal liver tissue, the hardened tissue and the HCC tissue are stained by immunostaining using the HCCR_1 according to the present invention, no antigen which reacts with HCCR4 is detected in the normal liver tissue and the cirrhotic tissue, and Antigens that reacted with HCCR-1 only in HCC tissues exhibited a dark brown appearance. Therefore, the diagnostic method and the kit according to the present invention can be very advantageously used for the early diagnosis of liver cancer and the diagnosis of small HCC, because they have high accuracy and reproducibility relative to the conventional App test, which is to be implemented by the following The invention is illustrated in detail, but is not intended to limit the scope of the invention to the particular embodiments described. <Example 1> Production of HCCR_1 polyclonal antibody<1-1> Production of HCCR-1 recombinant protein In order to produce a human proto-oncogene HCCR-丨 specific antibody as a tumor marker substance for diagnosis of liver cancer, Some human proto-oncogenes (for the detection of GenBank & (10) i〇nn〇AF195651, jID.N〇s: 123_473, amino acid 4mDN〇s: 39i5 placed on pET-32(+)
位而製備出 HCCR-lpET-32b㈩脱⑴載體。將該載體轉形到大腸桿g el2i(atcc Accession Νο· 4趣)之内並用i碰異丙基叫硫代半乳糖基-吼喃料 (IPTG)(Sigma Chemical Co.所製)以誘導出表現,產生具有融合到其上的 2〇Kda硫氧化還原蛋白(thioredoxin)(Trix)標籤(tag)蛋白質之35现HCCR1 融合蛋白質,且使用 01/27149)將其純化出。The HCCR-lpET-32b (de) off (1) vector was prepared. The vector was transformed into the large intestine rod g el2i (atcc Accession Νο. 4) and induced by i to hit isopropyl called thiogalactosyl-purine (IPTG) (manufactured by Sigma Chemical Co.) to induce It was shown that 35 of the current HCCR1 fusion protein with 2〇Kda thioredoxin (Trix) tag protein fused thereto was produced and purified using 01/27149).
His-Hind套組(Novagen所製)(國際專利公報w〇 對純化出的重組蛋自進行免疫點潰,確定其中含有 15 1290226 大里的約35kDa之融合蛋白質(圖i)。使用經如此離析和純化出的船 重組蛋白作為抗顧於抗原_抗體結合反應巾以賴和分析可產生用於小鼠 免疫化的單株抗體之融合瘤細胞系。 <1·2>兔子之免疫化The His-Hind kit (manufactured by Novagen) (International Patent Publications) was used to immunofuse the purified recombinant egg to determine that it contained 15 1290226 Dali of a fusion protein of approximately 35 kDa (Fig. i). The purified recombinant protein of the ship is used as a fusion tumor cell line which can produce a monoclonal antibody for mouse immunization against the antigen-antibody binding reaction towel. <1·2> Immuneization of rabbit
使用七個月大的雄紐西蘭白兔作為產生實施例〇中所製備的HCH 重組蛋白特細罐· _物。將纽蛋自(篇微統升)和 F_d s完全佐.igma Chemical c〇)以等量混合且乳化。將各〇 %毫升 的礼液她肖_發地接種顺子胸朗八健。於_雜接種之後, 以-星期之間隔進行加強注射,免疫化所用抗原係使用加仙心不完全佐 劑(Sigma所製者)予以乳化且使用與原發性接種相同的方式於u個星期之 期間接種該乳液。 <1_3>蛋黃抗艘(IgY)之離析 以-星期之間隔從耳靜脈採集錢樣品並從樣品離析出血清且在用於各種 實驗測量之翁膝耽。血清中抗體特異性之評定顯示丨該抗體只與人 類原-致癌基因HCCR-1蛋白質特異地反應。 <實施例2>HCCR_1單株抗體之產生 <2-1>小鼠的免疫化 為了得到可以產生HCOM單株抗體的融合瘤細胞系所需的免疫化小鼠, 乃將實施例叫舛所製的HCCW重組蛋白,Freund,s完全佐劑⑶興 Chemical Co.所製)和F麵d,s不完全侧(Gibc〇所製)予以混合。將所得混 合物經由《岐_ 5_6錄紙的祕/e小鼠_,如麟該小鼠以 兩個星期f·她匕次。要細生之時,就在最後—次加強之 16 1290226 前,以ELISA法測量小鼠血清的抗體滴定價。只有具有大於陰性對照組別 5倍的抗體滴定度之小鼠材加強。於加強之三天後,進行細胞融合檢驗。 <2-2>細胞融合(融合瘤(hybridoma))檢驗 用為要融合的母細胞之小鼠血漿骨髓細胞瘤細胞(myeloma cells)係得自the American Type of Culture Collection (ATCC),USA 的 SP_2 細胞系,且係在 融合檢驗的一星期前細胞展現出指數生長曲線之時才用於細胞融合。使該 細胞系置於含有10%胎牛血清的DMEM培養液内生長。 於最後一次免疫化三天之後,以活體組織檢查鉗從Balb/c小鼠切取出脾細 胞並將該脾細胞置於沒有血清的DMEM培養液内以製備脾細胞懸浮液。然 後,使所得溶液靜置於室溫下使結締組織沉著下來且隨後移除。將脾細胞 懸浮液以l,〇〇〇r.p.m·離心3分鐘,接著將脾細胞暴露於含有Tris 20.6克/ 升和NH4C1,8.3克/升的Tris_NH4Cl溶液以溶解紅血球細胞,接著用培養液 經由以1,000 rpm離心3分鐘於以洗滌3次,藉此將細胞數目到約1 X 1〇8 細胞/毫升。此外,從小鼠的眶靜脈叢採集1.5立方厘米的血液用為隨後實 驗中的陽性對照組。 將SP-2血漿骨髓細胞瘤細胞與脾細胞以1 : 1〇之比例混合,然後使用5〇% (w/v)聚乙二醇 1500 予以融合(Boerhinger Mannheim Co·,Germany)。為了 只篩選出融合瘤細胞,將細胞融合結果產生的細胞懸浮於包括15%胎牛血 清且含有50 μΜ次黃嘌呤(hypoxanthine),0.4 μΜ胺基喋呤(aminopterin)和 16μΜ胸腺核苷溶液的HAT溶液之Waymouth培養液中,且分配於96-洞微 滴定板(Coming Co” USA所製)(100微升/洞)。將融合瘤細胞置於5% C02 保溫箱内在37°C胞核溼度條件下培養。 17 1290226 <2-3>產生單株抗艘的融合瘤細胞系之篩選 為了篩選融合瘤細胞,乃以間接酵素聯結免疫吸著檢定(ELISA)法篩選可與 HCCR-1蛋白質抗原特異地反應之細胞。換言之,係以ELISA讀取器測量 吸光度以篩選會產生可高度結合到經純化的HCCR-1蛋白質的抗體之融合 瘤細胞系,且經由限制性稀釋將融合瘤細胞稀釋以使篩選出的成為單株, 藉此建立由從一早細胞增生出的細胞所構成的細胞系。為了從實施例<2_2> 所製備的融合瘤細胞組中篩選出只與人類原-致癌基因HCCR_1重組蛋白抗 原特異地反應之融合瘤細胞,乃於細胞融合之後的第10天,只從具有增生 細胞的洞中取出培養液上澄液且針對從實施例^-^製備的大腸桿菌轉形 體所離析和純化出的HCCR-1重組蛋白抗原施以ELISA。 詳細說來,為了壓制非特異性免疫反應,乃對96-洞板(Falc〇n c〇·,USA)施 加1%脫之牛乳-PBS且使彼靜置於室溫下丨小時,再將培養好的Hcc細胞 (ATCC,USA)以2 X 1〇5細胞/洞的量加到每一洞内。於每一洞中加入別微 升的融合瘤細胞培養液上澄液之後,在4〇c下誘發進行抗原抗體結合反應2 小時,使用PBS溶液洗務三次,且與作為繼發性抗體,用含有2% (w卵sa 的PBS緩衝液稀釋1/1〇,000過之山羊抗兔子职⑼興c〇,脱所製)在* °C下反應(100微升/洞)1小時。 其後’將100微升經由將10毫升ai M碟酸鹽緩衝棒樣酸碑5 〇),i毫克 3.3,,5.5,-四賈基聯苯胺(tmb) (Sigma c〇_,說所製)和2〇微升的娜過氧 化氫混合所得受質溶液加到每一洞内以誘發酵素反應。在室溫下維持該酵 素反應15分鐘,然後加入相同量的2 N破酸溶液以停止酵素反應。於· 不米下麟顏色顯絲度。單株抗體係經連續產生且制,個高度活化 18 1290226 的融口瘤細胞。於產SHCCRd蛋白質特異性抗體的融合瘤細胞中,只有 具有以ELISA測里時,比陰性對照組更大1〇倍的抗體滴定度之細胞才接受 進-步篩選’ _0_分析所篩選出的細胞所具特性。 之後’首絲騎產續HCCW纽蛋自抗料有高㈣雛結合活性 篩選出的細 的抗體之融合瘤細胞,且經由重複地實施相_程序數次對所 胞進-步騎,耻得财高齡HCCW纽蛋城紅與其特異 地反應之進-步篩選過的融合瘤細胞組。在使时髓細胞瘤細胞培養液作A seven-month-old male New Zealand white rabbit was used as the HCH recombinant protein ultrafine can be prepared in the Example. The egg is mixed and emulsified in equal amounts from the igma chemical c〇 and F_d s. The sputum of each 〇 % ml will be inoculated with a straight chest. After the vaccination, the booster injection was performed at intervals of -week, and the antigen used for the immunization was emulsified using the incomplete adjuvant (manufactured by Sigma) and used in the same manner as the primary vaccination for u weeks. The emulsion was inoculated during this period. <1_3> Isolation of egg yolk resistance (IgY) A money sample was taken from the ear vein at intervals of -week and serum was isolated from the sample and used for various experimental measurements. Evaluation of antibody specificity in serum revealed that the antibody specifically reacts only with the human proto-oncogene HCCR-1 protein. <Example 2> Production of HCCR_1 monoclonal antibody<2-1> Immunization of mice In order to obtain an immunized mouse required for a fusion tumor cell line capable of producing HCOM monoclonal antibodies, the example is called 舛The prepared HCCW recombinant protein, Freund, s complete adjuvant (3), manufactured by Chemical Co.) and the F-face d, s incomplete side (manufactured by Gibb) were mixed. The resulting mixture was passed through the 岐_5_6 recording paper/e mice _, such as Lin, the mice were licked twice a week. At the time of the birth, the antibody titer of the mouse serum was measured by ELISA before the last enhancement of 16 1290226. Only the mouse material having an antibody titer that was 5 times larger than the negative control group was reinforced. Three days after the boost, cell fusion assays were performed. <2-2> Cell fusion (hybridoma) test Mouse plasma myeloma cells used as mother cells to be fused are obtained from the American Type of Culture Collection (ATCC), USA The SP_2 cell line was used for cell fusion only when the cells exhibited an exponential growth curve a week before the fusion assay. This cell line was grown in DMEM medium containing 10% fetal bovine serum. Three days after the last immunization, spleen cells were excised from Balb/c mice by a biopsy forceps and the spleen cells were placed in serum-free DMEM culture medium to prepare a spleen cell suspension. Then, the resulting solution was allowed to stand at room temperature to cause the connective tissue to settle down and then removed. The spleen cell suspension was centrifuged at 1, rpm, for 3 minutes, and then the spleen cells were exposed to Tris_NH4Cl solution containing Tris 20.6 g/L and NH4C1, 8.3 g/L to dissolve red blood cells, followed by passage through the culture solution. Centrifuge at 1,000 rpm for 3 minutes to wash 3 times, thereby bringing the number of cells to about 1 X 1 〇 8 cells/ml. In addition, 1.5 cubic centimeters of blood was collected from the iliac venous plexus of the mice as a positive control group in the subsequent experiments. SP-2 plasma myeloid cell tumor cells were mixed with spleen cells in a ratio of 1:1, and then fused using 5 〇% (w/v) polyethylene glycol 1500 (Boerhinger Mannheim Co., Germany). In order to screen only the fusion tumor cells, the cells produced by the cell fusion were suspended in a solution containing 15% fetal calf serum and containing 50 μ of hypoxanthine, 0.4 μ of aminopterin and 16 μΜ of thymidine solution. HAT solution in Waymouth broth and dispensed in 96-well microtiter plate (manufactured by Coming Co. USA) (100 μL/well). The fusion tumor cells were placed in a 5% CO 2 incubator at 37 ° C. Incubation under humidity conditions. 17 1290226 <2-3> Screening of a single tumor-resistant fusion cell line to screen for fusion tumor cells, screening for HCCR-1 by indirect enzyme-linked immunosorbent assay (ELISA) A cell in which a protein antigen specifically reacts. In other words, an absorbance is measured by an ELISA reader to screen a fusion tumor cell line which produces an antibody highly bindable to the purified HCCR-1 protein, and the fusion tumor cell is subjected to restriction dilution. Diluted to make the selected individual plants, thereby establishing a cell line composed of cells proliferating from the early cells. In order to select only the fusion tumor cell group prepared from the example <2_2> The fusion cell of the human pro-oncogene HCCR_1 recombinant protein antigen specifically reacted on the 10th day after cell fusion, and only the culture solution was taken out from the well with the proliferating cells and prepared for the preparation from the example ^-^ The HCCR-1 recombinant protein antigen isolated and purified by the E. coli transformant was subjected to ELISA. In detail, in order to suppress the non-specific immune response, a 96-well plate (Falc〇nc〇·, USA) was applied. % of the milk was removed from the PBS and allowed to stand at room temperature for a few hours, and the cultured Hcc cells (ATCC, USA) were added to each well in an amount of 2 X 1 〇 5 cells/well. After adding a microliter of the mixed tumor cell culture medium to a well, the antigen-antibody binding reaction was induced for 2 hours at 4 ° C, washed three times with PBS solution, and used as a secondary antibody, containing 2 % (w) diluted egg PBS buffer 1/1 〇, 000 over goat anti-rabbit (9) 〇c〇, de-made) reacted at * °C (100 μl / hole) for 1 hour. Place 100 μl via 10 ml of ai M disc buffer buffer-like acid tablet 5 〇), i mg 3.3,, 5.5,-tetrakilylidene aniline ( Tmb) (Sigma c〇_, said to be prepared) and 2 〇 microliters of Na hydrogen peroxide mixed with the solution was added to each hole to induce fermentin reaction. Maintain the enzyme reaction at room temperature for 15 minutes, then The same amount of 2 N acid-breaking solution was added to stop the enzyme reaction. The color of the silk was reduced. The single-body anti-system was continuously produced and produced, and the highly activated 18 1290226 fused cells were produced. The SHCCRd protein was produced. Among the fusion tumor cells with specific antibodies, only cells with antibody titer one-fold more than the negative control group in the ELISA assay were subjected to the step-by-step screening of the characteristics of the cells selected by the analysis. . After that, 'the first silk rides the HCCW New Zealand egg self-resistance with high (four) chicks combined with the activity of the fine antibody fusion tumor cells, and through repeated implementation of the phase _ procedures several times on the cell into the step, shame Cai Gaoling HCCW New Egg City Red and its specifically responded to the step-filtered fusion tumor cell group. In the medullary cell culture medium
為陰性對照組之時,_顯示比陰_驗更大5倍的抗_定度的洞 中所含細胞。__的細胞_卜25 (Coming Co·,USA)以進行增生 。於此程序中,實施抗體滴定度測量3次以 上以在維持實質_的抗體滴定度之下選義中所含細胞,且將某些所選 殖的、、田胞貝τ存在_13〇C下。此外,於產生抗體的融合瘤細胞中,透過三次選 殖只師選出於維持高抗體滴定度之下仍然積極地增生之融合瘤細胞並予 以坧養以增生,且於其後用於單株抗體的生產中。At the time of the negative control group, _ showed cells that were 5 times larger than the yin-tested anti-determination hole. __ Cell_Bu 25 (Coming Co., USA) for proliferation. In this procedure, the antibody titer is measured more than 3 times to select the cells contained in the selection below the antibody titer that maintains the substantial amount, and some of the selected colonies are present in the _13〇C. under. In addition, in the antibody-producing fusion tumor cells, only three types of fusion tumor cells that are actively proliferating under high antibody titer are selected and maintained for proliferation by three colonizations, and are subsequently used for monoclonal antibodies. In production.
圖2閣示出為了篩選根據本發明的產生HCCIM單株抗體之融合瘤細胞系 而使用HCCR-1重組蛋白作為抗原所實施的咖八之結果。 k殖係以限制稀縣冑㈣。要選殖的齡瘤細胞之數目經調整使得在a 毫升培養液中包括有230個融合瘤細胞。其後,於96-洞板的36洞((U毫 升/洞)中各分配入5個細胞。將殘留的培養液(1毫升)與4毫升培養液混 合,且於剩餘的36洞(〇」毫升/洞)中分別配入i個細胞。將最後繼的培 養液(1.4毫升)與相同量的培養液混合且於24洞中分別配入〇 $個細胞(〇 ι ▲同)於14天之後於選殖株充足地生長之時,再度從培養液上澄液 19 1290226 鑑定出所產生的抗體之存在。將6陽性殖株轉移到一 24-洞板並予以谇養, 且最後鑑定所產生的抗體之存在。然後,只將陽性殖株轉移到一 25_毫升讲 養瓶中且料培養進行大規模生產。將某些陽性殖株貯存到維持在 液氮桶内。 <2-4>單株抗體的大量生產 為了使用實施例<2-2>所建立的融合瘤細胞系產生大量的對抗HccRq蛋白 · 質之單株抗體,乃於將融合瘤細胞經腹膜内注射到Balb/c小鼠體内的大約 · 10天之前,將0.5毫升的pristine (Sigma Co” USA所製)經由腹膜内投到每鲁 一小鼠體内,藉此增強產生腹膜内腫瘤的機率。隨後,將約1 X 1〇7融合瘤 細胞經由腹膜内注射以誘導腹水液之產生,且於其後,從每一小鼠的腹部 肥大處採集腹水液。如此產生的腹水液包括以高濃度生長的融合瘤細胞。 因此,將採集到的腹水液以10,000r.p.m·離心使融合瘤細胞沉著下來,藉 而只回收上澄液。於用來純化抗體蛋白質之前,將該上澄液貯存在_7〇〇c。 為了從腹水液純化抗體,乃進行蛋白質-A/G瓊脂糖管柱層析術(Pierce c〇., USA所製)與辛酸純化’藉此離析出免疫球蛋白(immun〇gi〇buiin)。最後產鲁 物,亦即,免疫球蛋白的濃度係經由使用Bio_Rad蛋白質微檢定套組(Bi〇_Rad Lab·,USA所製)以Bradford法予以測量。單株抗體免疫球蛋白的同型Fig. 2 shows the results of the application of HCCR-1 recombinant protein as an antigen for screening a fusion tumor cell line producing an HCCIM monoclonal antibody according to the present invention. k colony to limit the rare county 胄 (four). The number of aged tumor cells to be colonized was adjusted to include 230 fusion tumor cells in a ml of culture medium. Thereafter, 5 cells were dispensed into each of the 36 holes (U ml/hole) of the 96-well plate. The remaining culture solution (1 ml) was mixed with 4 ml of the culture solution, and the remaining 36 holes (〇) i cells were dispensed into the "ml/hole", and the last culture medium (1.4 ml) was mixed with the same amount of the culture medium, and 〇$ cells (〇ι ▲) were placed in the 24 holes respectively. After the day, when the selected plants were sufficiently grown, the presence of the produced antibodies was identified again from the culture solution, the liquid 19 1990226. The 6 positive plants were transferred to a 24-well plate and maintained, and finally the identification The presence of antibodies is generated. Then, only the positive colonies are transferred to a 25-ml vial and cultured for large-scale production. Some positive colonies are stored and maintained in liquid nitrogen tanks. 4> Mass production of monoclonal antibodies In order to use the fusion tumor cell line established in the example <2-2>, a large amount of monoclonal antibodies against HccRq protein was produced, and the fusion tumor cells were intraperitoneally injected into Balb. About 0.5 days before /c mice, 0.5 ml of pristine (Sigma Co" USA Transplanted intraperitoneally into each mouse, thereby increasing the probability of producing intraperitoneal tumors. Subsequently, about 1×1〇7 fusion tumor cells are injected intraperitoneally to induce the production of ascites fluid, and Thereafter, the ascites fluid was collected from the abdominal hypertrophy of each mouse. The ascites fluid thus produced included the fusion tumor cells grown at a high concentration. Therefore, the collected ascites fluid was centrifuged at 10,000 rpm to precipitate the fusion tumor cells. Only the supernatant is recovered. Before the antibody protein is purified, the supernatant is stored in _7〇〇c. In order to purify the antibody from the ascites, protein-A/G agarose column chromatography is performed. (Pierce c〇., USA) and octanoic acid purification 'to isolate immunoglobulin (immun〇gi〇buiin). Finally, the concentration of immunoglobulin, that is, the concentration of immunoglobulin is via the use of Bio_Rad protein micro-assay set The group (made by Bi〇_Rad Lab·, USA) was measured by the Bradford method. The isotype of monoclonal antibody immunoglobulin
(Isotypes)係利用一同型定型套組(Serotec Co.,England)使用血液吸著反應 (hemadsorbent reaction)予以測定。其結果,將單株抗體所離析出的同型鑑 定為IgG2b 〇 <實施例3>經由在HCCR-1單株抗體中經由免疫組織化學法染著以鑑定 HC 20 1290226 c組織 原-致癌基因HCCR-1的特異性,乃使 為了根據本發明確證單株抗體對人類 用該單株抗魏由免雜織化學轉縣蚊Hcc組織。 詳細言之,使職HCCR_1單株抗體以抗生物素蛋白_生物素·過氧化酶複合 物(avidin_bi〇tin-P觀idase c卿㈣(舰)法^ 常肝組織和HCC _。於操作處理過財賴要翁免疫_化學染著 的新鮮腫雜織’並將___置於液氮中快稍結,猶存於 下。 將從HCC患者切下的組織切成5微米厚度,於挪丙嗣中固定,且使其置 於0.5%過氧化氳/甲醇溶液内%分鐘以移除内源過氧化酶活性,接著用 TBS洗滌。此外,為了壓辦特異性免疫反應,將組織切片暴露於正常馬 血清(Vector Lab·,USA所製)3〇分鐘,並將作為第一抗體的稀釋單株抗體以 不同的濃度加到其中,隨後使其在代靜置24小時,接著用挪洗條。於 所得產物中加入生物素化馬抗-兔子IgG溶液(VectorLab.,USA所製)作為第 二抗體,在使其靜置約1小時。將所得產物再用TBS洗滌,並加入抗生物 素蛋白-生物素-過氧化酶複合物(Vector Lab·,USA所製)作為第三抗體,再使 其靜置40分鐘,接著充分地洗滌。將所得產物暴露於含有過氧化氳和 ΤΒΤ(3,3·二胺基聯苯胺·四鹽酸鹽)的受質溶液並以顯微鏡觀察顏色顯現。然 後’為了鑑疋可與HCC組織樣品内的抗體反應之細胞的發生率盘分布乃 用蘇木素(hematoxylin)免疫逆染著(counter-stained)組織切片且用光學顯' 鏡予以觀察。 其結果,經確定在HCC組織中含有比正常肝組織和HCC的原型之肝硬化 21 1290226 組織中遠較為大量的HCCR-丨蛋白質抗原(圖3)。 <實施例4>使用HCCR-1單株抗體進行的elisa 使用HCCR-1單株抗體按下述方式以eusa診斷肝癌。首先,用從融合瘤 細胞離析和純化所得HCCr]特異性抗體塗覆犯认板。第二步,用塗覆 在板洞上的抗體處理_檢體樣品並與之反應。第三步,傭在該檢體樣品 中的HCCR-1抗原_抗體結合之存在。 <4-1> HCCR-1單株抗禮塗覆 將HCCR-1蛋白質特異性單株抗體放置在%洞EUSA平底板中,用蓋子 蓋好且靜置於4°C下16至18小時使其塗覆。將經純化的單株抗體在〇·5 M 碳酸鹽緩衝液(pH 9·6)中稀制5微克/冑升之濃度且將1〇〇微升稀釋溶液 加到每-湖。作鱗敵者,係將沒減染蛋自f的正常小鼠 血π在石反酸鹽緩衝液中稀釋5〇〇_倍且分配到每一洞内(1〇〇微升/洞)。 然後’用洗滌緩衝液,亦即,含有〇 〇5% Tween 2〇的pBS⑽7 4成該板 的洞4次。其後’為了阻斷非特異性蛋白質結合部位,於每一洞中分配入 含有2% BSA阻斷溶液的PBS緩衝溶液&Η 7·4) (3〇〇微升/洞)且使其淨置 於37 C下2小時。接著’移除阻斷溶液並真空包裝以貯存於代下。 <4-2>於洞上所塗覆的抗艘舆檢艘樣品之間的反應 使用HCC和肝炎肝血清,非母體和母浙血清及正常肝血清作為檢體樣 。口°為了得雌止侧量賴的鮮鱗,乃經由將HCCR]重組蛋白以 刀別為G cfe微克/¾升(ng/ ),2G毫微克/毫升,4()毫微克/毫升,⑽毫微克 /¾升’ 16〇宅微克/毫升,32〇毫微克/毫升與_毫微克/毫升的不同濃度稀 釋而製備標準抗原浴液八、6'(];'〇、£、卩和0。 22 1290226 將刀別為100的個別檢體樣品分配到實施例<4-1>所準備的有塗覆著 HCCR-1單株抗體的每_洞中並在抓下反應4小時,接著用洗務緩衝液 洗;條4 -人。此處’係·上面所製備的鮮抗原溶刻^陽㈣照組且使 用正常小鼠血清作為陰性對照組。 <4-3>抗原-抗體結合之偵檢 將100微升辣根過氧化酶標記山羊抗·兔子IgG繼發性抗體的1〇,〇〇〇_倍稀釋 液加到每―;时,並使該板靜置於3rc下卜j、時,接著用洗雜衝液洗務 4次。隨後,將1毫克作為受質的33,,5·5,_四甲基聯苯胺(TMB)(sigmaC〇·, USA所製)溶解在10毫升檸檬酸鹽緩衝液碑5 〇)巾且於其中加入:微升的 35%過乳化氫,藉而製備出一受質溶液。將·微升所製備的受質溶液分配 到每-納且在室溫沒有暴朗光之下反應15分鐘。其後,加人⑽微升2 ΝΗΘ〇4溶液以停止反應且測量4〇5奈米吸光度。 對每-檢體樣品,HCCR_1抗原的吸光度係經由將塗覆著正常小鼠血清作 為陽f生對照組與塗覆著pBS作為陰性對照組的洞之總吸光度從塗覆著單株 抗體洞所得吸光度鱗的餘數。以_的方式,制鮮溶㈣吸光度值。 將鮮抗原溶液A和F所具吸献之_平均值定為截止值以供後面用來(Isotypes) were determined using a homotyped kit (Serotec Co., England) using a hemadsorbent reaction. As a result, the isotype isolated from the monoclonal antibody was identified as IgG2b 〇 <Example 3> by immunohistochemical staining in HCCR-1 monoclonal antibody to identify HC 20 1290226 c tissue proto-oncogene HCCR The specificity of -1 is such that in order to confirm according to the present invention, the monoclonal antibody against the human is used to control the Hcc tissue of the mosquito. Specifically, the HCCR_1 monoclonal antibody was treated with avidin-biotin/peroxidase complex (avidin_bi〇tin-P idase cqing (four) (ship) method ^ normal liver tissue and HCC _. After the money, I want to immunize _ chemically dyed fresh swollen weaving' and put ___ in liquid nitrogen to quickly knot, still underneath. Cut the tissue cut from HCC patients into 5 micron thickness, It was fixed in a propanil and placed in a 0.5% cerium peroxide/methanol solution for 1 minute to remove endogenous peroxidase activity, followed by washing with TBS. In addition, in order to press a specific immune response, the tissue was sliced. Exposure to normal horse serum (manufactured by Vector Lab., USA) for 3 minutes, and the diluted monoclonal antibody as the first antibody was added thereto at different concentrations, and then allowed to stand for 24 hours, followed by The obtained product was added with a biotinylated horse anti-rabbit IgG solution (manufactured by VectorLab., USA) as a second antibody, and allowed to stand for about 1 hour. The obtained product was further washed with TBS, and an anti-antigen was added thereto. Biotin-biotin-peroxidase complex (manufactured by Vector Lab., USA) as the first The antibody was allowed to stand for another 40 minutes, followed by thorough washing. The obtained product was exposed to a solution containing cerium peroxide and cerium (3,3·diaminobenzidine tetrahydrochloride) and observed under a microscope. The color appears. Then 'in view of the incidence of cells that can react with antibodies in HCC tissue samples, the disk distribution was counter-stained with hematoxylin and visualized with an optical microscope. As a result, it was confirmed that the HCC tissue contained a much larger amount of HCCR-丨 protein antigen in the liver cirrhosis 21 1290226 than the normal liver tissue and the prototype of HCC (Fig. 3). <Example 4> Using HCCR-1 alone Elisa by antibody The liver cancer was diagnosed by eusa using HCCR-1 monoclonal antibody as follows. First, the antibody was coated with the HCCr]-specific antibody isolated and purified from the fusion tumor cells. The second step was coated with The antibody on the plate is treated with a sample of the sample and reacted with it. In the third step, the HCCR-1 antigen-antibody binding in the sample is present. <4-1> HCCR-1 Coating a specific strain of HCCR-1 protein The body was placed in a % hole EUSA flat bottom plate, covered with a lid and allowed to stand at 16 ° C for 16 to 18 hours. The purified monoclonal antibody was in 〇·5 M carbonate buffer (pH 9). · 6) Dilute the concentration of 5 μg / liter and add 1 〇〇 microliter of diluted solution to each lake. For scaly enemies, the normal mouse blood will not be subtracted from f. Dilute 5 〇〇 times in the acid salt buffer and dispense into each well (1 μL/well). Then, the plate was drilled 4 times with a washing buffer, i.e., pBS (10) 7 4 containing 〇 5% Tween 2 。. Thereafter, in order to block the non-specific protein binding site, a PBS buffer solution containing 2% BSA blocking solution & Η 7·4) (3 μL/well) was dispensed into each well and allowed to The net was placed at 37 C for 2 hours. The blocking solution was then removed and vacuum packed for storage. <4-2> Reaction between the samples of the anti-cavity test specimens coated on the holes HCC and hepatitis liver serum, non-maternal and maternal serum and normal liver serum were used as samples. In order to obtain the fresh scale of the female side, the recombinant protein of HCCR] is labeled as G cfe microgram / 3⁄4 liter (ng / ), 2G ng / ml, 4 () ng / ml, (10) Prepare a standard antigen bath with nanograms/3⁄4 liters of '16 〇μg/ml, 32 〇 ng/ml diluted with _ nanograms/ml. Prepare a standard antigen bath VIII, 6'(]; '〇, £, 卩, and 0 22 1290226 An individual sample sample having a knives of 100 was assigned to each of the holes prepared with the HCCR-1 monoclonal antibody prepared in Example <4-1> and subjected to a reaction for 4 hours, followed by a reaction for 4 hours. Washed with washing buffer; strip 4 - human. Here, the fresh antigen prepared in the above is immersed in the yang (4) group and the normal mouse serum is used as the negative control group. <4-3> Antigen-antibody Combined detection of 100 μl of horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody 1 〇, 〇〇〇 _ dilution was added to each ―; and the plate was placed under 3rc Then, it was washed four times with the washing liquid. Then, 1 mg was dissolved as 33,5,5,4-tetramethylbenzidine (TMB) (manufactured by Sigma Co., USA). In 10 ml of citric acid The salt buffer was used to add a microliter of 35% per-emulsified hydrogen to prepare a substrate solution. The substrate solution prepared by microliter was dispensed to per-nano and reacted for 15 minutes at room temperature without violent light. Thereafter, (10) microliters of a 2 ΝΗΘ〇 4 solution was added to stop the reaction and the absorbance of 4 〇 5 nm was measured. For each sample, the absorbance of the HCCR_1 antigen was obtained by coating the antibody body of the individual antibody by the total absorbance of the hole coated with normal mouse serum as the positive control group and the hole coated with pBS as the negative control group. The remainder of the absorbance scale. In the way of _, the fresh (four) absorbance value is prepared. The average value of the fresh antigen solutions A and F is set as the cutoff value for later use.
判疋檢體為陽性或陰性檢體。亦即,當一檢體的吸光度大於鮮抗原溶液F 的吸光度之時,將雜體稀釋且制量其吸光度。將具有高於截止值的吸 光度之檢體鑑疋為陽性,且將具有低於截止值的吸光度之檢體鑑定為。 此處&準抗原浴液A的平均吸光度必須大於或等於議G且小於或等於 0·2〇0。標準抗原溶液F的平均吸光度必須大於或等於i•且小於或等於 3.000。 23 1290226 截止值經從個HCCR_1鮮抗聽_備的鮮鱗败為ω微克/毫 升。根據此截止值,比較_檢體的吸光度值以欺鮮為陽性或陰性。 肝癌組,肝炎患者組,母體組和正常組的比較結果都顯示於圖如至扣之中。 <實施例5>使用HCCR-1單株抗體以ELISA確定HCC診斷效率 為了確定使用HCCR-1單株抗體的ELISA診斷套組和方法的肝癌診斷效 率,乃使用實施例4中所述HCCR-1特異性抗體進行ELISA測量及使用傳 統α -胎蛋白(AFP)檢驗進行測量以供比較。afp水平係使用市面上得自as Bio International,France 的 ELSA2_AFP 套組予以測量的。 為了根據診斷結果判定檢體為陽性或陰性,將AFP的截止值設定為2〇毫微 克/毫升且將HCCR-1的截止值定為1〇微克/毫升,此係得自標準曲線者。 於每一 HCC組、肝炎組和正常母體組中在AFP陽性與HCCR-1陽性之間 的反應性差異都與使用McNemar檢驗所得數據相比較。對於肝癌組與正常 組’ HCCR-1敏感度,特異性,偽陽性/陰性比例都是在95%信任區間測量 的。 將肝癌組根據2厘米的腫瘤尺寸分成兩組。將每一個別組之間的HCCR-1 陽性反應性差異與用Fishery精確試驗(Fisherfs exact test)所測數據相比 較。將每一組的AFP陽性反應性與HCCR-1陽性反應性之間的差異與 McNemar試驗所測數據相比較。於所有統計分析中使用SAS變異值6.12 且使用 0.05 之有效水平(significance level) (SAS/STAT Software: Changes andThe specimen is judged to be a positive or negative specimen. That is, when the absorbance of a sample is larger than the absorbance of the fresh antigen solution F, the hybrid is diluted and the absorbance is measured. A sample having an absorbance higher than the cutoff value is regarded as positive, and a sample having an absorbance lower than the cutoff value is identified as. Here, the average absorbance of the & quasi-antigen bath A must be greater than or equal to the G and less than or equal to 0·2〇0. The average absorbance of the standard antigen solution F must be greater than or equal to i• and less than or equal to 3.000. 23 1290226 The cutoff value is reduced to ωμg/ml from the fresh scale of HCCR_1. Based on this cutoff value, the absorbance value of the sample is compared to be positive or negative by bullying. The comparison results of the liver cancer group, the hepatitis patient group, the maternal group and the normal group are shown in the figure. <Example 5> Determination of HCC diagnostic efficiency by ELISA using HCCR-1 monoclonal antibody In order to determine the diagnostic efficiency of liver cancer using the ELISA diagnostic kit and method of HCCR-1 monoclonal antibody, the HCCR-described in Example 4 was used. 1 Specific antibodies were subjected to ELISA measurements and measured using conventional alpha-fetoprotein (AFP) assays for comparison. The afp levels were measured using the ELSA2_AFP kit available from as Bio International, France. In order to determine whether the sample is positive or negative based on the diagnosis result, the cutoff value of AFP is set to 2 〇 nanogram/ml and the cutoff value of HCCR-1 is set to 1 〇 microgram/ml, which is obtained from the standard curve. The difference in reactivity between AFP positive and HCCR-1 positive in each HCC group, hepatitis group, and normal maternal group was compared with data obtained using the McNemar test. For the liver cancer group and the normal group, the HCCR-1 sensitivity, specificity, and pseudo-positive/negative ratio were measured in the 95% confidence interval. The liver cancer group was divided into two groups according to the tumor size of 2 cm. The difference in HCCR-1 positive reactivity between each individual group was compared to the data measured by the Fishery exact test (Fisherfs exact test). The difference between AFP positive reactivity and HCCR-1 positive reactivity for each group was compared to the data measured by the McNemar test. Use SAS variability of 6.12 in all statistical analyses and use a 0.05 level of significance (SAS/STAT Software: Changes and
Enhancements through Release 6· 12. Cary,NC: SAS Institute,1997) o <5-l> AFP舆HCCR-1套組對於HCC患者組的診斷正確性 表1顯示出HCCR-1與AFP套組對於由97個肝癌患者所構成的HCC組的 24 1290226 診斷正確性之比較結果。該等結果顯示出根據Η··〗的診斷正確性為 94.9% ’其明顯地較高於根據AFP所得結果,7〇 1% (chi—ΜΛ2=19 2,步卜 P<0扁卜McNemar試驗)。特別者’ 29個肝癌患者之中經频為娜陰 性的27個(93.1%)經正確地診斷為Ηα:ΙΜ陽性,而68個肝癌患者中的3 個患者(4.4%)經診斷為AFP陽性,亦即經診斷為HccRq陰性。亦即, 診斷係不正確者。 表1 AF^ - 陽性 陰性 合計 HCCR-1 陽性 65 27 92 (94.9%) 陰性 3 一 2 5 ___ 合計 68(70.1%) 29 197(ϊδ〇%) 4-25 AFP與HCCR-1套組對於正常母艘組的診斷正碟性 從表2明顯可知,於由72個女人所構成的正常母體組中,根據jjccn and AFP兩套組的反應性結果都是陽性者。亦即,72的正常母體女人全部都是 陽性,而只有11個母體女人(15·3%)為HCCR-1陽性,導致明顯的差異 (cliiJV[A2=61,dfH,ρ<〇·00(π,McNemar 試驗)。因此,可以說 AFP 肝 癌診斷對於正常母體組係實質地無意義者。不過,可以確定者根據HCCR-1 的診斷可顯著地減少診斷錯誤率。 表2 AFP 陽性 陰性 合計 25 1290226 HCCR-1 陽性 11 0 11(15.3%) 陰性 61 0 61 合計 72(100%) 0 72(100%) <5-3>AFP與HCCR-1套組對於肝炎患者組的診斷正確性 表3顯示出AFP與HCCR-1套組對於由72個肝炎患者組所得診斷結果。 52個肝炎患者中有2個(3.9%)經根據HCCR-1而錯誤地診斷尨叶疬弟、去, 而52個肝炎患者中有11個(21.2%)經根棱HCCR-1而錯誤地診斷麁吁滤棗 ±(chi—MA2=7.3636,dfH,P=〇.〇〇67,McNemar 試驗)。特別者,u 個經 錯誤地診斷為肝癌患者中有1〇個(90.9%)經診斷為陽性。亦即,HCCR-1診 斷比AFP診斷遠較為準確。 表3 AFP ------ 陽性 陰性 合計 HCCR-1 陽性 11 0 11(15.3%) —— 陰性 61 0 6Ϊ ·~— 合計 72(100%) 0 72(100%) ' <5_4>HCCR_1套組作為肝癌讀套組的利用性之肯定 如表4和5情示者,97個肝絲者和⑵個正常人接钱驗。本發明 HCCM套組得敏感度為94外且其特異性%篇,偽陽性和偽陰性分別為 ^和4.4%。而且,其整體診斷正確性為似,確定本發明配咖套組 非常可用為診斷工具。 26 1290226 表4Enhancements through Release 6· 12. Cary, NC: SAS Institute, 1997) o <5-l> AFP舆HCCR-1 kit for the correctness of the diagnosis of the HCC patient group Table 1 shows the HCCR-1 and AFP kits for Comparison of the diagnostic accuracy of 24 1290226 of the HCC group consisting of 97 liver cancer patients. These results show that the diagnostic accuracy according to Η·· is 94.9% 'which is significantly higher than the results obtained according to AFP, 7〇1% (chi-ΜΛ2=19 2, step P<0 flat Bu McNemar test ). In particular, 27 of the 29 liver cancer patients (93.1%) were correctly diagnosed as Ηα:ΙΜ positive, and 3 of 68 liver cancer patients (4.4%) were diagnosed as AFP positive. , that is, diagnosed as HccRq negative. That is, the diagnosis is incorrect. Table 1 AF^ - positive negative total HCCR-1 positive 65 27 92 (94.9%) negative 3 - 2 5 ___ total 68 (70.1%) 29 197 (ϊδ〇%) 4-25 AFP and HCCR-1 kits for normal The diagnosis of the mother ship group is clearly evident from Table 2. In the normal mother group consisting of 72 women, the reactivity results of the two groups according to jjccn and AFP were positive. That is, 72 normal mother women are all positive, while only 11 maternal women (15.3%) are HCCR-1 positive, resulting in significant differences (cliiJV[A2=61, dfH, ρ<〇·00( π, McNemar test). Therefore, it can be said that the diagnosis of AFP liver cancer is substantially meaningless to the normal maternal group. However, it can be determined that the diagnosis error rate can be significantly reduced according to the diagnosis of HCCR-1. Table 2 AFP positive negative total 25 1290226 HCCR-1 positive 11 0 11 (15.3%) negative 61 0 61 total 72 (100%) 0 72 (100%) <5-3> AFP and HCCR-1 kit for the diagnosis of hepatitis patients 3 shows the results of the AFP and HCCR-1 kits for the 72 patients with hepatitis. 2 out of 52 hepatitis patients (3.9%) were mistakenly diagnosed according to HCCR-1, and 52 Eleven of the patients with hepatitis (21.2%) were mistakenly diagnosed by the root-edge HCCR-1 (麁-MA2=7.3636, dfH, P=〇.〇〇67, McNemar test). Special, u One (90.9%) of the patients who were diagnosed as having liver cancer were diagnosed as positive. That is, the diagnosis of HCCR-1 is far more accurate than the diagnosis of AFP. Indeed. Table 3 AFP ------ positive negative total HCCR-1 positive 11 0 11 (15.3%) —— negative 61 0 6 Ϊ ·~- total 72 (100%) 0 72 (100%) ' <5_4> The HCCR_1 kit as a liver cancer reading kit is affirmative as shown in Tables 4 and 5, 97 liver silkers and (2) normal people receiving money. The HCCM kit of the present invention has a sensitivity of 94 and its The % specificity, false positive and false negative were ^ and 4.4%, respectively. Moreover, the overall diagnostic accuracy was similar, and it was determined that the catering set of the present invention is very useful as a diagnostic tool. 26 1290226 Table 4
-------- 測量 —·~~~— ----— Value (%) —^ 〜....................................一 95%信任區間 敏感度 92/97=94.9 ^^^_ 90·5 〜99.3 特異性 ~~~~~~~_ 109/121=90.1 —^_ 84.8^95.4 偽陽性 S --- 12/104=11.5 ^^_ 5.4 〜17.6 偽陰性 —~~~--- 5/114=4.4 0.6 〜8.2 陽性預測水平 -------- 92/104=88.5 82.3 〜94.6 陰性預測水平 ~~~------- 109/114=95.6 91.8 〜99.4 正確性 -------- (92+109)/218=92.2 88.6 〜95.8 <5_5>依110^腫瘤尺寸的HCCR-1套組診斷正確性~ 〜--------- Measurement —·~~~— ----— Value (%) —^ ~....................... ............. A 95% confidence interval sensitivity 92/97=94.9 ^^^_ 90·5 ~99.3 Specificity ~~~~~~~~ 109/121=90.1 — ^_ 84.8^95.4 False Positive S --- 12/104=11.5 ^^_ 5.4 ~17.6 False Negative -~~~--- 5/114=4.4 0.6 ~8.2 Positive Prediction Level -------- 92/104=88.5 82.3 ~94.6 Negative prediction level ~~~------- 109/114=95.6 91.8 ~99.4 Correctness-------- (92+109)/218=92.2 88.6 ~ 95.8 <5_5> Diagnosis of correctness of HCCR-1 kit according to 110^ tumor size~~-
表6顯示出對於85個具有不小於2厘米的腫瘤之肝癌患者與…固具有不 大於2厘米的軸之賴患麵實施的檢驗之賴正雜 〇 . 珂於具有不小 於2厘麵_尺寸之患者有約72.9%之AFP·增加率,㈣於且有不 大於__瘤尺寸之患者有約·之辦陽性增加率,可推測出^ 腫瘤尺爾蝴輯脚綱加# ^ hccri 21 為基底的為、、。果在所有腫叙寸上_示出超過·㈣性。不過, 27 1290226 HCCR-1和AFP兩種診斷套組在腫瘤尺寸上沒有顯示出明顯的差異。 於所有的腫瘤尺寸中,HCCR-1診斷套組顯示出比AFP套組明顯較高的陽 性反應性(Ρ=〇·〇25,ρ=〇·〇〇ι)。於不大於2厘米的腫瘤組織中,HCCR_1陽 性者比AFP陽性者較高41.7%。於不小於2厘米的腫瘤組織中,HCCR-1 陽性者比AFP陽性者較高22·4%。此意味著根據本發明的HCCR-1診斷套 組可以有利地用來診斷小尺寸的腫瘤。 表6 陽性反應性(%) 腫瘤尺寸§ AFP tlCCR-1 <2厘米 6/12=50.0 11/12=91.7* 22厘米 62/85=72.9 81/85=95.31 — §Ρ>0·05,依χ2檢定和Fisher,s精確試驗在ΑΡΡ與HCCR4之間根據腫瘤 尺寸的陽性反應性差異。 *Ρ=0·025,依McNemar試驗在AFP與HCCR-1之間根據不大於2厘米腫瘤 尺寸的陽性反應性差異。 28 1 Ρ=0·001,依McNemar試驗在AFP與HCCR-1之間根據不小於2厘米腫 瘤尺寸的陽性反應性差異。 工業應用性 如上文所述者,由於根據本發明的含有人類原_致癌基因HCCRq特異性抗 體的肝癌讀套組為使用患者血清的免疫診斷玉具,具有比傳統婚檢驗 工具較高的正雜和再離,因此其可有利地用於賴的早期診斷與小 HCC的診斷。Table 6 shows the test for the liver cancer patients with 85 tumors having a tumor of not less than 2 cm and the axis of the shaft having a shaft of not more than 2 cm. 珂 Having a size of not less than 2 centimeters _ size The patient has an AFP increase rate of about 72.9%, and (4) the patient has a positive rate of increase in the size of the patient who is not larger than the __ tumor size. It can be inferred that the tumor is a ruler and the foot is plus #^hccri 21 The base is . If it is on all swollen inches, it shows more than (four) sex. However, 27 1290226 HCCR-1 and AFP diagnostic kits did not show significant differences in tumor size. Among all tumor sizes, the HCCR-1 diagnostic kit showed significantly higher positive reactivity than the AFP kit (Ρ=〇·〇25, ρ=〇·〇〇ι). In tumor tissues of no more than 2 cm, HCCR_1 positive patients were 41.7% higher than AFP positive patients. In tumor tissues of not less than 2 cm, HCCR-1 positive patients were 22.4% higher than AFP positive patients. This means that the HCCR-1 diagnostic kit according to the present invention can be advantageously used to diagnose small-sized tumors. Table 6 Positive reactivity (%) Tumor size § AFP tlCCR-1 <2 cm 6/12=50.0 11/12=91.7* 22 cm 62/85=72.9 81/85=95.31 — §Ρ>0·05, The Dependent 2 assay and the Fisher, s exact test showed a positive reactivity difference between sputum and HCCR4 based on tumor size. *Ρ=0·025, according to the McNemar test, the positive reactivity difference between AFP and HCCR-1 according to tumor size of no more than 2 cm. 28 1 Ρ=0·001, according to the McNemar test, the positive reactivity difference between AFP and HCCR-1 according to the tumor size of not less than 2 cm. Industrial Applicability As described above, since the liver cancer reading kit containing the human proto-oncogene HCCRq-specific antibody according to the present invention is an immunodiagnostic jade using patient serum, it has a higher quality than the conventional marriage test tool. And re-dissociation, so it can be advantageously used for the early diagnosis of Lai and the diagnosis of small HCC.
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